PROJECT SUMMARY The initiation of an immune response requires specialized antigen presenting cells to present antigen alongside co- stimulatory molecules. Activation of toll like receptors (TLRs) is often involved in expression of these co-stimulatory molecules. TLR pathways detect pathogen-associated molecular patterns (PAMPs), which are common in viruses and bacteria but not humans. Dozens of TLRs have now been identified in humans and animal models. TLR agonists (TLRas) have been investigated as vaccine adjuvants due to their ability activate TLRs and upregulate certain co-stimulatory molecules. Mammalian TLR3, TLR9, TLR13, and TLR7/8 are located within endosomes where they can sense and bind endocytosed microbial and nucleic acid based PAMPs to drive immune response. Recent studies show activating multiple TLRs with their respective TLR agonists (TLRas) increases the efficacy of vaccines for infectious diseases and cancer. Therefore delivering programmable combinations of TLRas with disease relevant antigen may be exploited to create a strong antigen-specific immune responses tuned for particular pathogens. While traditional co-delivery methods often rely on biomaterials, biomaterials themselves often elicit immune responses that can confound vaccine design because the carrier changes the response to the other vaccines components (e.g., antigen, TLRa). Our lab has previously developed immune polyelectrolyte multilayers (iPEMs), a modular platform exploiting electrostatic interactions to create carrier-free capsules composed entirely of antigen and single TLRa. This strategy allows co-delivery of tunable signals without extraneous components, providing a means to isolate the impact of specific TLRas in polarizing immunity. This proposal will use the iPEM platform to reveal how co-delivering different combinations and relative ratios of TLRas changes the magnitude and type of the resulting immune response during generation of antigen-specific immunity. In preliminary experiments I have shown iPEMs deliver TLRa cargo to endosomes resulting in time-dependent immune activation in vitro. Our lab has further shown that iPEMs composed of a model antigen and a single TLRa enhance antigen-specific immune response compared to soluble delivery of the same components. Additionally, iPEMs can be synthesized from defined combinations and ratios of different classes of TLRas. This proposal will build on these data to 1) Elucidate the effect of iPEM composition (combinations of TLRas) on the in vitro trafficking and immune function, 2) Test if iPEM composition can be used to program TLR signaling in vivo, and 3) Confirm that activating multiple TLRas using iPEMs improves immune response and survival during a mouse model of melanoma. This model will be used as a functional test bed since recent studies revealed that treatment with two TLRas increased survival in a mouse model of melanoma. The knowledge gained from this proposal will show how co-delivery of different combinations of TLRas can control the type and level of the resulting immune response. This can be applied to any vaccines currently in the clinic for infectious diseases and cancer immunotherapies.